Dihydrolipoamide dehydrogenase (E3) is a common flavoprotein component of the alpha-keto acid dehydrogenase complexes involved in the oxidation of pyruvate, alpha-ketoglutarate and branched-chain alpha-keto acids in the mitochondria. E3-deficient patients show developmental delay and severe neurologic disabilities. The long term goal of this proposal is to continue to enhance our understanding of the structure-function relationship of human E3 at the level of protein and transcriptional regulation of the gene in normal and disease states. Based on our recent findings we have expanded the scope of this proposal. Five specific aims of this proposal are: (i) to investigate the structure-function relationship of normal and site-specifically mutated human E3s by measuring kinetic and spectral characteristics, (ii) to determine the crystal structure of normal and mutant human E3s, (iii) to characterize the transcriptional regulation of the promoter region of the human E3 gene by hormones in cultured cells and developmental and tissue-specific regulation in transgenic mice, (iv) to characterize the molecular basis for E3 deficiency in affected patients, and (v) to regulate the expression of human E3 in cells to investigate possible metabolic consequences. We have constructed a prokaryotic expression vector which is capable of producing large quantities of recombinant human E3 required for structural studies. Initial characterization of the human E3 promoter indicates that this promoter has several unique characteristics for a "housekeeping" gene and is subject to hormonal manipulations. Mutation analysis in E3- deficient patients has already enhanced our understanding of the structure-function relationship and is likely to contribute significantly to the proposed studies. Our multifaceted collaborative approach to studying human E3 at the protein and gene levels should further contribute to our knowledge of the structure-function relationship of E3 protein and transcriptional regulation of the E3 gene.